Page 239 - Fundamentals of Light Microscopy and Electronic Imaging

222 CONFOCAL LASER SCANNING MICROSCOPY

Apply offset Apply gain
Original signal (add negative voltage) (amplify signal)

Amplitude (volts)

Distance
(a) (b) (c)
Figure 12-12
Adjustment of gain and offset. Gain and offset are electronic controls used to stretch the
available photon signals from an object to fill the dynamic range of the detector in order to
include the maximum number of gray levels, from white to black, in the captured image.
Offset adds a voltage (positive or negative) to the output signal so that the lowest signals just
approach the threshold for detection on the PMT (black level). Gain amplifies the signal by
multiplying the output voltage from the PMT by a constant factor prior to its digitization at the
analogue-to-digital converter and is increased until the maximum signal values just approach
saturation. Offset should be applied first before adjusting the gain.

PMT. After digitization in the computer, the photon signal is displayed as shades of gray
ranging from black (no signal) to white (saturating signal) on the computer monitor.
10
12
PMTs with a dynamic range of 10 or 12 bits have 2 (1024) or 2 (4096) gray levels,
respectively, which is also the number of gray levels in the respective image files in the
computer. Setting the gain and offset of the PMT should not be confused with adjusting
the contrast and brightness of the image during image processing. Processing can
stretch existing pixel values to fill the black-to-white display range on the monitor, but
can never create new gray levels. Thus, when a poorly captured image containing only
50 out of a possible 4000 gray levels is stretched from black to white during image pro-
cessing, the resulting image looks grainy. Sometimes it is not possible to fill all of the
gray levels during an acquisition, but ordinarily this should be the goal. We will discuss
the gray-level ranges of images in Chapters 13 through 16.
Offset is an electronic adjustment that adds a positive or negative voltage to the sig-
nal so that a selected background signal corresponds to a PMT output of 0 volts
(black). Gain is an electronic adjustment that amplifies the input signal by a voltage
multiplication process that causes it to be assigned to a higher gray-level value. Gener-
ally, offset should be set first, followed by adjustment of the gain. Increasing the gain
beyond its optimal setting can make an image look somewhat grainy, but it is sometimes
desirable to capture the maximum number of gray levels, even if the gain must be
increased somewhat to do so. In practice, these adjustments are often made using a spe-
cial color display function to depict pixel values on the monitor. In one commonly used
function, the saturating pixels are shown in red, black-level pixels are shown in blue,
and all intermediate gray levels are shown in shades of gray. When properly adjusted, a
few red and blue pixels will be seen in the image, indicating that the full dynamic range